U.S. patent number 10,272,126 [Application Number 15/216,134] was granted by the patent office on 2019-04-30 for nanoparticle-stabilized microcapsules, dispersions comprising nanoparticle-stabilized microcapsules, and method for the treatment of bacterial biofilms.
This patent grant is currently assigned to THE UNIVERSITY OF MASSACHUSETTS. The grantee listed for this patent is The University of Massachusetts. Invention is credited to Bradley Duncan, Xiaoning Li, Vincent M. Rotello.
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United States Patent |
10,272,126 |
Duncan , et al. |
April 30, 2019 |
Nanoparticle-stabilized microcapsules, dispersions comprising
nanoparticle-stabilized microcapsules, and method for the treatment
of bacterial biofilms
Abstract
A stabilized microcapsule includes a liquid hydrophobic core
including an essential oil and an aromatic monoaldehyde, and a
shell encapsulating the core, the shell including a plurality of
amino-functionalized inorganic nanoparticles. Also disclosed is a
dispersion including a plurality of the stabilized microcapsules.
The microcapsules and dispersions can be particularly useful for
treating a bacterial biofilm.
Inventors: |
Duncan; Bradley (Andover,
MA), Li; Xiaoning (Hillsboro, OR), Rotello; Vincent
M. (Amherst, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Massachusetts |
Boston |
MA |
US |
|
|
Assignee: |
THE UNIVERSITY OF MASSACHUSETTS
(Boston, MA)
|
Family
ID: |
58156778 |
Appl.
No.: |
15/216,134 |
Filed: |
July 21, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170049113 A1 |
Feb 23, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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62208114 |
Aug 21, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N
25/28 (20130101); A61K 31/12 (20130101); A01N
25/04 (20130101); A61K 36/534 (20130101); A61K
47/52 (20170801); A61K 9/501 (20130101); A61L
2/0082 (20130101); A01N 35/02 (20130101); A01N
25/28 (20130101); A01N 27/00 (20130101); A01N
35/02 (20130101); A01N 37/40 (20130101); A01N
49/00 (20130101); A01N 65/00 (20130101); A01N
65/10 (20130101); A01N 65/22 (20130101); A01N
65/24 (20130101); A01N 65/28 (20130101); A01N
35/02 (20130101); A01N 27/00 (20130101); A01N
37/40 (20130101); A01N 49/00 (20130101); A01N
65/00 (20130101); A01N 65/10 (20130101); A01N
65/22 (20130101); A01N 65/24 (20130101); A01N
65/28 (20130101); A01N 25/04 (20130101); A01N
27/00 (20130101); A01N 35/02 (20130101); A01N
37/40 (20130101); A01N 49/00 (20130101); A01N
65/00 (20130101); A01N 65/10 (20130101); A01N
65/22 (20130101); A01N 65/24 (20130101); A01N
65/28 (20130101); Y02A 50/30 (20180101) |
Current International
Class: |
A01N
65/22 (20090101); A61K 9/50 (20060101); A01N
25/28 (20060101); A01N 35/02 (20060101); A01N
25/22 (20060101); A61K 47/52 (20170101); A01N
31/04 (20060101); A01N 25/04 (20060101); A61L
2/00 (20060101); A61K 36/534 (20060101); A61K
9/107 (20060101); A61K 31/12 (20060101); A61L
2/18 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2015016368 |
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Feb 2015 |
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JP |
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2009063257 |
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May 2009 |
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WO |
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|
Primary Examiner: Phan; Doan T
Attorney, Agent or Firm: Cantor Colburn LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH &
DEVELOPMENT
This invention was made with government support under Grant Number
EB014277 awarded by the National Institute of Health (NIH) and
under Grant Number CMMI-1025020 awarded by the National Science
Foundation (NSF) Center for Hierarchical Manufacturing. The
government has certain rights in the invention.
Claims
The invention claimed is:
1. A stabilized microcapsule comprising, a single liquid
hydrophobic core comprising an essential oil and an aromatic
monoaldehyde; and a single shell encapsulating the core, the shell
consisting of a plurality of amino-functionalized inorganic
nanoparticles having one or more amino functional groups directly
covalently bound to the surface of the inorganic nanoparticles; one
or more amino functional groups covalently bound to the surface of
the inorganic nanoparticles by a linker comprising a C.sub.1-12
alkyl group, a C.sub.6-20 aryl group, or an alkylene oxide group
between the amino functional group and the surface; or combination
thereof; wherein the essential oil is selected from the group
consisting of peppermint oil, oregano oil, thymol, menthol, methyl
salicylate, eucalyptol, carvacrol, camphor, anethole, carvone,
eugenol, isoeugenol, osimen, n-decyl alcohol, citronel,
a-salpineol, methyl acetate, citronellyl acetate, methyl eugenol,
cineol, ethyl linalaol, safrola vanillin, spearmint oil, lemon oil,
orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil,
laurel oil, cedar leaf oil, clove oil, cilantro oil, coriander oil,
and a combination thereof; and wherein the aromatic monoaldehyde is
cinnamaldehyde.
2. A stabilized microcapsule comprising, a single liquid
hydrophobic core comprising an essential oil and an aromatic
monoaldehyde; and a single shell encapsulating the core, the shell
consisting of a plurality of functionalized inorganic nanoparticles
having one or more amino functional groups directly covalently
bound to the surface of the inorganic nanoparticles; one or more
amino functional groups covalently bound to the surface of the
inorganic nanoparticles by a linker comprising a C.sub.1-12 alkyl
group, a C.sub.6-20 aryl group, or an alkylene oxide group between
the amino functional group and the surface; a reaction product
formed from reaction of an amino-functionalized inorganic
nanoparticle and the aromatic monoaldehyde, or a combination
thereof; wherein the essential oil is selected from the group
consisting of peppermint oil, oregano oil, thymol, menthol, methyl
salicylate, eucalyptol, carvacrol, camphor, anethole, carvone,
eugenol, isoeugenol, osimen, n-decyl alcohol, citronel,
a-salpineol, methyl acetate, citronellyl acetate, methyl eugenol,
cineol, ethyl linalaol, safrola vanillin, spearmint oil, lemon oil,
orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil,
laurel oil, cedar leaf oil, clove oil, cilantro oil, coriander oil,
and a combination thereof; and wherein the aromatic monoaldehyde is
cinnamaldehyde.
3. The microcapsule of claim 1, wherein the inorganic nanoparticles
are metal nanoparticles, metal oxide nanoparticles, or a
combination thereof, and wherein the nanoparticles have one or more
dimensions of less than 1000 nanometers.
4. The microcapsule of claim 1, wherein the inorganic nanoparticles
comprise silica, titanium dioxide, or a combination thereof.
5. The microcapsule of claim 1, wherein the inorganic nanoparticles
have an average diameter of 100 to 250 nanometers.
6. The microcapsule of claim 1, wherein the essential oil comprises
peppermint oil.
7. The microcapsule of claim 1, comprising 0.01 to 10 volume
percent of the aromatic monoaldehyde, based on the total volume of
the essential oil and the aromatic monoaldehyde.
8. The microcapsule of claim 1, comprising 1 to 10 weight percent
of the inorganic nanoparticles, based on the total weight of the
microcapsule.
9. The microcapsule of claim 1, wherein the microcapsule has an
average diameter of 1 to 20 micrometers.
10. The microcapsule of claim 1, wherein the essential oil
comprises peppermint oil; the aromatic monoaldehyde is
cinnamaldehyde; the microcapsule comprises 1 to 5 volume percent
cinnamaldehyde, based on the total volume of the essential oil and
the aromatic monoaldehyde; the inorganic nanoparticles are silica
nanoparticles having an average diameter of 140 to 160 nanometers;
and the microcapsule has an average diameter of 4 to 9
micrometers.
11. The microcapsule of claim 1, comprising, based on the total
weight of the microcapsule, 90 to 99 weight percent of the liquid
hydrophobic core; and 1 to 10 weight percent of the shell
encapsulating the core; wherein the liquid hydrophobic core
comprises, based on the total weight of the core, 90 to 99 weight
percent peppermint oil; and 1 to 10 weight percent
cinnamaldehyde.
12. A method of treating a bacterial biofilm the method comprising,
contacting a dispersion comprising a plurality of the stabilized
microcapsule according to claim 1 with a bacterial biofilm.
13. The method of claim 12, wherein the stabilized microcapsules
are dispersed in an aqueous solution.
14. The method of claim 12, wherein each microcapsule comprises a
shell consisting of silica nanoparticles; and the silica is present
in an amount of less than or equal to 3 weight percent, based on
the total weight of the dispersion.
15. The method of claim 12, wherein the bacterial biofilm comprises
Escherichia coli, Pseudomonas bacteria, Staphylococcal bacteria,
Enterobacteriaceae bacteria, Streptococcus bacteria, Haemophilus
influenzae, Leptospira interrogans, Legionella bacteria, or a
combination thereof.
16. The method of claim 12, wherein the contacting is in the
presence of a host cell, wherein the dispersion is non-toxic to the
host cell.
Description
BACKGROUND OF THE INVENTION
Bacterial biofilms are highly resilient microbial assemblies that
are difficult to eradicate. See, e.g., Costerton, J. W.; Stewart,
P. S.; Greenburg, E. P. Bacterial Biofilms: A Common Cause of
Persistent Infections. Science 1999, 284, 1318-1322. These robust
biofilms frequently occur on synthetic implants and indwelling
medical devices including urinary catheters, arthro-prostheses, and
dental implants. See, e.g., Lindsay, D.; von Holy, A. Bacterial
Biofilms within the Clinical Setting: What Healthcare Professionals
Should Know. J. Hosp. Infect. 2006, 64, 313-325; Costerton, J. W.;
Montanaro, L.; Arciola, C. R. Biofilm in Implant Infections: Its
Production and Regulation. Int. J. Artif. Organs 2005, 28,
1062-1068; Busscher, H. J.; Rinastiti, M.; Siswomihardjo, W.; van
der Mei, H. C. Biofilm Formation on Dental Restorative and Implant
Materials. J. Dent. Res. 2010, 89, 657-665. Biofilm proliferation
can also occur on dead or living tissues, leading to endocarditis,
otitis media, and chronic wounds. See, e.g., Costerton, W.; Veeh,
R.; Shirtliff, M.; Pasmore, M.; Post, C.; Ehrlich, G. The
Application of Biofilm Science to the Study and Control of Chronic
Bacterial Infections. J. Clin. Invest. 2003, 112, 1466-1477;
Ehrlich, G.; Veeh, R.; Wang, X.; Costerton, J. W.; Hayes, J. D.;
Hu, F. Z.; Daigle, B. J.; Ehrlich, M. D.; Post, J. C. Mucosal
Biofilm Formation on Middle-Ear Mucosa in the Chinchilla Model of
Otitis Media. JAMA 2002, 287, 1710; James, G. A; Swogger, E.;
Wolcott, R.; Pulcini, E. deLancey; Secor, P.; Sestrich, J.;
Costerton, J. W.; Stewart, P. S. Biofilms in Chronic Wounds. Wound
Repair Regen. 2007, 16, 37-44. The persistent infections and their
concomitant diseases are challenging to treat, as biofilms develop
a high resistance to host immune responses and the extracellular
polymeric substances limit antibiotic penetration into biofilms.
See, e.g., Stewart, P. S.; Costerton, J. W. Antibiotic Resistance
of Bacteria in Biofilms. Lancet 2001, 358, 135-138; Szomolay, B.;
Klapper, I.; Dockery, J.; Stewart, P. S. Adaptive Responses to
Antimicrobial Agents in Biofilms. Environ. Microbiol. 2005, 7,
1186-1191. Current techniques to remove biofilms on man-made
surfaces include disinfecting the surface with bleach or other
caustic agents. See, e.g., Marion-Ferey, K.; Pasmore, M.; Stoodley,
P.; Wilson, S.; Husson, G. P.; Costerton, J. W. Biofilm Removal
from Silicone Tubing: An Assessment of the Efficacy of Dialysis
Machine Decontamination Procedures Using an in Vitro Model. J.
Hosp. Infect. 2003, 53, 64-71. Biofilms in biomedical contexts are
very challenging, with therapies based on excising infected tissues
combined with long-term antibiotic therapy, incurring high health
care costs and low patient compliance due to the invasive
treatment. See, e.g., Lynch, A. S.; Robertson, G. T. Bacterial and
Fungal Biofilm Infections. Annu. Rev. Med. 2008, 59, 415-428. This
issue is exacerbated by the exponential rise in antibiotic
resistant bacteria. See, e.g., Levy, S. B.; Marshall, B.
Antibacterial Resistance Worldwide: Causes, Challenges and
Responses. Nat. Med. 2004, 10, S122-S129.
Phytochemicals have emerged as a promising alternative to
traditional antimicrobials to treat antibiotic resistant bacteria.
See, e.g., Kalemba, D.; Kunicka, A. Antibacterial and Antifungal
Properties of Essential Oils. Curr. Med. Chem. 2003, 10, 813-829;
Hemaiswarya, S.; Kruthiventi, A. K.; Doble, M. Synergism between
Natural Products and Antibiotics against Infectious Diseases.
Phytomedicine 2008, 15, 639-652. These essential oils and natural
compounds are of particular interest as "green" antimicrobial
agents due to their low-cost, biocompatibility, and potential
anti-biofilm properties. See, e.g., Burt, S. Essential Oils: Their
Antibacterial Properties and Potential Applications in Foods--a
Review. Int. J. Food Microbiol. 2004, 94, 223-253; Kavanaugh, N.
L.; Ribbeck, K. Selected Antimicrobial Essential Oils Eradicate
Pseudomonas Spp. and Staphylococcus Aureus Biofilms. Appl. Environ.
Microbiol. 2012, 78, 4057-4061; Nostro, A.; Sudano Roccaro, A.;
Bisignano, G.; Marino, A.; Cannatelli, M. A; Pizzimenti, F. C.;
Cioni, P. L.; Procopio, F.; Blanco, A. R. Effects of Oregano,
Carvacrol and Thymol on Staphylococcus Aureus and Staphylococcus
Epidermidis Biofilms. J. Med. Microbiol. 2007, 56, 519-523. The
generally poor aqueous solubility and stability of these oils has
substantially limited their widespread application. See, e.g.,
Chen, H.; Davidson, P. M.; Zhong, Q. Impacts of Sample Preparation
Methods on Solubility and Antilisterial Characteristics of
Essential Oil Components in Milk. Appl. Environ. Microbiol. 2014,
80, 907-916. Engineering nanomaterials provides a potential
platform to prevent payload degradation and to tune molecular
interactions with bacteria. See, e.g., Carpenter, A. W.; Worley, B.
V; Slomberg, D. L.; Schoenfisch, M. H. Dual Action Antimicrobials:
Nitric Oxide Release from Quaternary Ammonium-Functionalized Silica
Nanoparticles. Biomacromolecules 2012, 13, 3334-3342; Zhu, X.;
Radovic-Moreno, A. F.; Wu, J.; Langer, R.; Shi, J. Nanomedicine in
the Management of Microbial Infection--Overview and Perspectives.
Nano Today 2014, 9, 478-498; Radovic-Moreno, A. F.; Lu, T. K.;
Puscasu, V. a; Yoon, C. J.; Langer, R.; Farokhzad, O. C. Surface
Charge-Switching Polymeric Nanoparticles for Bacterial Cell
Wall-Targeted Delivery of Antibiotics. ACS Nano 2012, 6, 4279-4287;
Goswami, S.; Thiyagarajan, D.; Das, G.; Ramesh, A. Biocompatible
Nanocarrier Fortified with a Dipyridinium-Based Amphiphile for
Eradication of Biofilm. ACS Appl. Mater. Interfaces 2014, 6,
16384-16394. Previous reports have shown that encapsulating
essential oils into surfactant-stabilized colloidal delivery
vehicles improves their aqueous stability and increases the
antimicrobial activity of small molecule payloads. See, e.g.,
Chang, Y.; McLandsborough, L.; McClements, D. J. Physicochemical
Properties and Antimicrobial Efficacy of Carvacrol Nanoemulsions
Formed by Spontaneous Emulsification. J. Agric. Food Chem. 2013,
61, 8906-8913; Liang, R.; Xu, S.; Shoemaker, C. F.; Li, Y.; Zhong,
F.; Huang, Q. Physical and Antimicrobial Properties of Peppermint
Oil Nanoemulsions. J. Agric. Food Chem. 2012, 60, 7548-7555; Gomes,
C.; Moreira, R. G.; Castell-Perez, E. Poly
(DL-Lactide-Co-Glycolide) (PLGA) Nanoparticles with Entrapped
Trans-Cinnamaldehyde and Eugenol for Antimicrobial Delivery
Applications. J. Food Sci. 2011, 76, N16-N24. However, these
carriers often induce adverse hemolytic or irritating effects
restricting their compatibility with biological tissues. See, e.g.,
Shalel, S.; Streichman, S.; Marmur, A. The Mechanism of Hemolysis
by Surfactants: Effect of Solution Dispersion. J. Colloid Interface
Sci. 2002, 252, 66-76; Wilhelm, K.-P.; Freitag, G.; Wolff, H. H.
Surfactant-Induced Skin Irritation and Skin Repair. J. Am. Acad.
Dermatol. 1994, 30, 944-949. Pickering emulsions provide an
analogous route to encapsulate hydrophobic molecules within a
self-assembled colloidal shell that is highly resistant to
coalescence. See, e.g., Ramsden, W. Separation of Solids in the
Surface-Layers of Solutions and "Suspensions" (Observations on
Surface-Membranes, Bubbles, Emulsions, and Mechanical
Coagulation).--Preliminary Account. Proc. R. Soc. London 1903, 72,
156-164; Pickering, S. U. Emulsions. J. Chem. Soc. Trans. 1907, 91,
2001. The multivalent nanoparticles embedded at the oil/water
interface can also be post-functionalized to create structurally
diverse carriers not achievable when using surfactant stabilized
emulsions. See, e.g., Binks, B. P. Particles as
Surfactants--Similarities and Differences. Curr. Opin. Colloid
Interface Sci. 2002, 7, 21-41; Ghouchi Eskandar, N.; Simovic, S.;
Prestidge, C. A. Nanoparticle Coated Submicron Emulsions: Sustained
in-Vitro Release and Improved Dermal Delivery of All-Trans-Retinol.
Pharm. Res. 2009, 26, 1764-1775.
BRIEF SUMMARY OF EMBODIMENTS OF THE INVENTION
One embodiment is a stabilized microcapsule comprising a liquid
hydrophobic core comprising an essential oil and an aromatic
monoaldehyde; and a shell encapsulating the core, the shell
comprising a plurality of amino-functionalized inorganic
nanoparticles.
Another embodiment is a stabilized microcapsule comprising a liquid
hydrophobic core comprising an essential oil; and a shell
encapsulating the core, the shell comprising a plurality of
amino-functionalized inorganic nanoparticles, wherein the
amino-functionalized inorganic nanoparticles further comprise a
reaction product covalently bound to a surface of the nanoparticle,
wherein the reaction product is formed from reaction of an
amino-functionalized inorganic nanoparticle and an aromatic
monoaldehyde.
Another embodiment is a dispersion comprising a plurality of
stabilized microcapsules.
Another embodiment is a method of treating a bacterial biofilm, the
method comprising contacting the dispersion with a bacterial
biofilm.
These and other embodiments are described in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
The following Figures are exemplary embodiments.
FIG. 1 shows a transmission electron micrograph (TEM) of silica
nanoparticles. The nanoparticles had an average diameter of
152.+-.15 nanometers (nm). Inset is a histogram of the measured
nanoparticle diameters. Scale bar is 500 nm.
FIG. 2 shows average diameter of the silica nanoparticles in MilliQ
H.sub.2O as determined by dynamic light scattering (DLS).
FIG. 3 shows zeta potential of the silica nanoparticles in 5
millimolar (mM) sodium phosphate buffer adjusted to pH 7.4.
FIG. 4 shows a schematic depiction of the strategy used to generate
antimicrobial capsules (a), and subsequent use for treatment of a
biofilm (b).
FIG. 5a shows an optical microscopy image of capsules generated
using an aqueous phase comprised of 0.3 wt. % silica (average
diameter=6.1.+-.3.0 micrometers (.mu.m)). Inset is the histogram of
the capsule diameter measurement. Scale bar is 50 .mu.m.
FIG. 5b shows an optical microscopy image of capsules generated
using an aqueous phase comprised of 0.6 wt. % silica (average
diameter=7.1.+-.3.0 .mu.m). Inset is the histogram of the capsule
diameter measurement. Scale bar is 50 .mu.m.
FIG. 5c shows an optical microscopy image of capsules generated
using an aqueous phase comprised of 0.9 wt. % silica (average
diameter=6.7.+-.2.1 .mu.m). Inset is the histogram of the capsule
diameter measurement. Scale bar is 50 .mu.m.
FIG. 5d shows an optical microscopy image of capsules generated
using an aqueous phase comprised of 1.2 wt. % silica (average
diameter=6.5.+-.1.7 .mu.m). Inset is the histogram of the capsule
diameter measurement. Scale bar is 50 .mu.m.
FIG. 5e shows an optical microscopy image of capsules generated
using an aqueous phase comprised of 1.5 wt. % silica (average
diameter=6.6.+-.2.1 .mu.m). Inset is the histogram of the capsule
diameter measurement. Scale bar is 50 .mu.m.
FIG. 5f shows a box plot of the capsules at various silica loadings
demonstrating capsule dispersity minimizes with increasing wt. % of
silica nanoparticles.
FIG. 6a shows optical an microscopy image of peppermint oil based
capsules ("P-Cap").
FIG. 6b shows an optical microscopy image of capsules containing 5%
v/v of cinnamaldehyde dissolved in peppermint oil ("CP-Cap").
FIG. 7a shows confocal micrographs of P-Cap. The nanoparticles'
cores are labeled with fluorescein and the oil phases are loaded
with Nile red. Scale bars are 20 .mu.m.
FIG. 7b shows confocal micrographs of CP-Cap. The nanoparticles'
cores are labeled with fluorescein and the oil phases are loaded
with Nile red. Scale bars are 20 .mu.m.
FIG. 7c shows X-ray photoelectron spectra (XPS) showing N 1s core
levels arising from SiO.sub.2 NPs and CP-Cap.
FIG. 7d shows water contact angles of silica nanoparticles
following incubation with varying concentrations of
cinnamaldehyde.
FIG. 8 shows XPS spectra of amino-functionalized SiO.sub.2 NPs,
CP-Caps, and Schiff base.
FIG. 9 shows attenuated total reflection Fourier transform infrared
spectroscopy (ATR-FTIR) characterization of cinnamaldehyde, the
cinnamaldehyde-silane Schiff base, the silica nanoparticles, and
CP-Caps after freeze drying.
FIG. 10 shows representative digital images of water droplets used
to determine contact angles.
FIG. 11 shows representative 3D projections of confocal image
stacks of 1 day-old E. coli DH5.alpha. biofilm after 3 hrs
treatment with a) CP-Cap containing FITC-labeled SiO2 NP, b) P-Cap
containing FITC-labeled SiO.sub.2 NP, and c) FITC-labeled SiO.sub.2
NP at 20% (v/v % of 2% emulsion) concentration. Upper panels are
projection at 247.degree. angle turning along Y axis and lower
panels are at 270.degree. angle turning along Y axis. Scale bars
are 20 .mu.m.
FIG. 12a shows the viability of 1 day-old P. aeruginosa (CD-1006)
biofilms after 3 hours treatment with CP-Cap, P-Cap, SiO.sub.2 NP,
and peppermint oil at different emulsion concentrations (v/v % of
2% emulsion). The data are average of triplicates and the error
bars indicate the standard deviations.
FIG. 12b shows the viability of 1 day-old E. coli DH5.alpha.
biofilms after 3 hours treatment with CP-Cap, P-Cap, SiO.sub.2 NP,
and peppermint oil at different emulsion concentrations (v/v % of
2% emulsion). The data are average of triplicates and the error
bars indicate the standard deviations.
FIG. 12c shows the viability of 1 day-old S. aureus (CD-489)
biofilms after 3 hours treatment with CP-Cap, P-Cap, SiO.sub.2 NP,
and peppermint oil at different emulsion concentrations (v/v % of
2% emulsion). The data are average of triplicates and the error
bars indicate the standard deviations.
FIG. 12d shows the viability of 1 day-old E. cloacae complex
(CD-1412) biofilms after 3 hours treatment with CP-Cap, P-Cap,
SiO.sub.2 NP, and peppermint oil at different emulsion
concentrations (v/v % of 2% emulsion). The data are average of
triplicates and the error bars indicate the standard
deviations.
FIG. 13a shows the viability of 3T3 fibroblast cells and E. coli
biofilms in the co-culture model after 3 hours treatment with
CP-Cap at different emulsion concentrations (v/v % of 2% emulsion).
Scatters and lines represent 3T3 fibroblast cell viability. Bars
represent log.sub.10 of colony forming units in biofilms. The data
are average of triplicates and the error bars indicate the standard
deviations.
FIG. 13b shows the viability of 3T3 fibroblast cells and E. coli
biofilms in the co-culture model after 3 hours treatment with P-Cap
at different emulsion concentrations (v/v % of 2% emulsion).
Scatters and lines represent 3T3 fibroblast cell viability. Bars
represent log.sub.10 of colony forming units in biofilms. The data
are average of triplicates and the error bars indicate the standard
deviations.
FIG. 13c shows the viability of 3T3 fibroblast cells and E. coli
biofilms in the co-culture model after 3 hours treatment with
SiO.sub.2 NP at different emulsion concentrations (v/v % of 2%
emulsion). Scatters and lines represent 3T3 fibroblast cell
viability. Bars represent log.sub.10 of colony forming units in
biofilms. The data are average of triplicates and the error bars
indicate the standard deviations.
FIG. 13d shows the viability of 3T3 fibroblast cells and E. coli
biofilms in the co-culture model after 3 hours treatment with
peppermint oil at different emulsion concentrations (v/v % of 2%
emulsion). Scatters and lines represent 3T3 fibroblast cell
viability. Bars represent log.sub.10 of colony forming units in
biofilms. The data are average of triplicates and the error bars
indicate the standard deviations.
DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is the fabrication of a multifunctional essential
oil-based Pickering emulsion. The emulsions can be particularly
useful for the treatment of bacterial biofilms. The self-assembly
strategy relies on hydrophobic phytochemicals playing both
antimicrobial and structural roles for the drug delivery vehicle.
Droplets comprising an essential oil, such as peppermint oil,
provide the main hydrophobic core template for nanoparticle
assembly into microcapsules. Dissolved cinnamaldehyde plays a dual
role within the oil core by covalently reacting with the
nanoparticles at the interface to modify the shell of the capsules
from within and acting as a potent antimicrobial agent once
delivered into the biofilm. These microcapsules effectively
eradicate both laboratory and pathogenic biofilms. The inclusion of
cinnamaldehyde also enhanced fibroblast proliferation promoting
therapeutic behavior of the capsules as demonstrated in an in vitro
co-culture model. See, e.g., Takasao, N.; Tsuji-Naito, K.;
Ishikura, S.; Tamura, A.; Akagawa, M. Cinnamon Extract Promotes
Type I Collagen Biosynthesis via Activation of IGF-I Signaling in
Human Dermal Fibroblasts. J. Agric. Food Chem. 2012, 60, 1193-1200.
This work presents a versatile colloidal strategy for
multicomponent essential oil formulations with potential use as a
general topical antimicrobial and disinfectant
Accordingly, one aspect of the present disclosure is a stabilized
microcapsule. The stabilized microcapsule has a core-shell
morphology. In some embodiments, the stabilized microcapsule
comprises a liquid hydrophobic core comprising an essential oil and
an aromatic monoaldehyde. In some embodiments, the stabilized
microcapsule comprises a liquid hydrophobic core comprising an
essential oil (e.g., no free, unreacted aromatic monoaldehyde is
present). The liquid hydrophobic core is generally a liquid at
25.degree. C. The essential oil can be a naturally occurring
compound (e.g., derived from a plant). Essential oils, as used
herein, are volatile aromatic oils which can be synthetic or
derived from plants (e.g., flowers, buds, seeds, leaves, twigs,
bark, herbs, wood, fruits, roots, and the like) by a physical
method (e.g., distillation, expression, fermentation, or
extraction). Essential oils usually carry the odor or flavor of the
plant from which they are obtained. The essential oil is preferably
an oil having antimicrobial properties. In some embodiments, the
essential oil can be selected from, for example, peppermint oil,
oregano oil, thymol, menthol, methyl salicylate (wintergreen oil),
eucalyptol, carvacrol, camphor, anethole, carvone, eugenol,
isoeugenol, limonene, osimen, n-decyl alcohol, citronel,
a-salpineol, methyl acetate, citronellyl acetate, methyl eugenol,
cineol, linalool, ethyl linalaol, safrola vanillin, spearmint oil,
lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil,
pimento oil, laurel oil, cedar leaf oil, clove oil, cilantro oil,
coriander oil, or a combination thereof. In some embodiments, the
essential oil is selected from peppermint oil, cilantro oil,
coriander oil, cinnamon oil, oregano oil, rosemary oil, sage oil,
clove oil, thyme oil, or a combination thereof. In some
embodiments, the essential oil is peppermint oil. In some
embodiments, the essential oil is present in an amount of 90 to 99
or 95 to 99 weight percent, based on the total weight of the core.
In some embodiments, the essential oil is present in an amount of
90 to 99.9, or 90 to 99, or 95 to 99 volume percent, based on the
total volume of the core.
In addition to the essential oil, the liquid hydrophobic core of
the microcapsule can include an aromatic monoaldehyde. The aromatic
monoaldehyde is miscible with the essential oil. In some
embodiments, the aromatic monoaldehyde is cinnamaldehyde.
Cinnamaldehyde (also known as cinnamic aldehyde or
3-phenyl-2-propenal) is a main component of cassia oil and cinnamon
bark oil. Cinnamaldehyde is also a major antimicrobial compound in
cinnamon. In some embodiments, the aromatic monoaldehyde is present
in an amount of 0.01 to 10, or 0.1 to 10, or 1 to 10, or 1 to 5
volume percent, based on the total volume of the essential oil and
the aromatic monoaldehyde (i.e., the total volume of the liquid
hydrophobic core). In some embodiments, the aromatic monoaldehyde
is present in an amount of 0.01 to 10, or 0.1 to 10, or 1 to 10, or
1 to 5 weight percent, based on the total weight of the core.
In some embodiments, the total weight of the core (e.g., the weight
of the essential oil and the aromatic monoaldehyde) constitutes 90
to 99, or 92 to 98, or 94 to 96 weight percent of the total weight
of the microcapsule.
In addition to the liquid hydrophobic core, the microcapsule of the
present disclosure comprises a shell encapsulating the core. The
shell comprises a plurality of amino-functionalized inorganic
nanoparticles. In some embodiments, the shell comprises a monolayer
of amino-functionalized inorganic nanoparticles. The inorganic
nanoparticles can be metal nanoparticles, metal alloy
nanoparticles, metal oxide nanoparticles, or a combination thereof.
The nanoparticles generally have one or more dimensions of less
than 1000 nanometers, for example less than 500 nanometers, for
example, less than 250 nanometers. In some embodiments, the
nanoparticles are spherical nanoparticles. In some embodiments, the
nanoparticles are silica nanoparticles, silicon nanoparticles, gold
nanoparticles, titanium oxide nanoparticles, and the like, or a
combination thereof. In some embodiments, the nanoparticles
comprise silica, titanium dioxide, or a combination thereof. In
some embodiments, the inorganic nanoparticles comprise silica, for
example the inorganic nanoparticles can be silica nanoparticles. In
some embodiments, the inorganic nanoparticles have an average
diameter of 75 to 500 nanometers, or 100 to 250 nanometers, or 100
to 200 nanometers, or 125 to 175 nanometers, or 135 to 170
nanometers, or 140 to 160 nanometers.
The inorganic nanoparticles are amino-functionalized nanoparticles.
"Amino-functionalized" means that one or more amino functional
groups are present on the surface of the nanoparticle. In some
embodiments, the amino functional group is covalently bound to the
surface of the nanoparticles. In some embodiments, the
amino-functionalized nanoparticles include a linker between the
amino group and the surface of the nanoparticle, for example a
linker comprising a C.sub.1-12 alkyl group, a C.sub.6-20 aryl
group, or an alkylene oxide group. In some embodiments, the amino
group can be directly bound to the surface of the nanoparticle
(i.e., no linker is present). In some embodiments, for example when
the nanoparticle is a metal nanoparticle, the amino functional
group can be covalently bound to a ligand that is associated with
the surface of the nanoparticle though ligand-type interactions.
The chemical structure of the ligand can be selected by those
skilled in the art with regards to the specific ligand-type
interaction and strength of those interactions with the
nanoparticle. The ligand-type interaction can be any suitable
bonding or non-bonding interaction. Ligand type interactions
comprise high affinity or low affinity site-specific type
interactions, non-bonded electrostatic interactions such as
electropositive or electronegative type or van der Waals repulsive
and attractive forces, ionic bonds, hydrogen bonds, coordination
bonds, or a combination thereof. For example, a gold nanoparticle
can interact with a thiol-functionalized ligand including the amino
functional group. In some embodiments, the amino group is a primary
amino group.
In some embodiments, when the nanoparticles comprise silica, the
surface of the silica nanoparticle can be functionalized through
reaction of the silica with an aminosilane, for example aminopropyl
trimethoxysilane, aminopropyl triethoxysilane, or the like. The
silane can react with the silica to provide primary amine groups
covalently attached to the nanoparticle surface. An extensive range
of silanes exist and can be selected by those skilled in the art to
provide the desired amino-functionalized nanoparticle.
In some embodiments, an amino-functionalized nanoparticle of the
microcapsule shell can react with the aromatic monoaldehyde present
in the core to provide a corresponding reaction product. For
example, the portion of the surface of the nanoparticle that is
exposed to and in contact with the liquid hydrophobic core can
react with one or more aromatic monoaldehydes to provide a
nanoparticle wherein at least a portion of the surface of the
nanoparticle comprises the reaction product of the
amino-functionalized nanoparticle and the aromatic monoaldehyde,
thus promoting the formation of Janus-type particles, defined as a
particle having two distinct regions (e.g., hemispheres) of
functionality. The aromatic monoaldehyde can be linked to the
surface of the nanoparticle by formation of the corresponding
aromatic imine (--C.dbd.N--). Thus, in some embodiments, the shell
of the microcapsule can comprise amino-functionalized
nanoparticles, inorganic nanoparticles covalently bound to a
reaction product formed from reaction of an amino group and an
aromatic monoaldehyde, or a combination thereof. In some
embodiments, the inorganic nanoparticles can include both an amino
functional group and the corresponding imino-linked reaction
product bound to the surface of the nanoparticle, preferably
wherein substantially all of the imino-linked reaction product is
in contact with the core of the microcapsule. Stated another way,
the microcapsule can include a shell comprising inorganic
nanoparticles, wherein each nanoparticle has two regions of
functionality: the first region comprises an amino functional
group, and the second region comprises a reaction product formed
from reaction of the amino functional group and an aromatic
monoaldehyde. In an embodiment, the second region of each
nanoparticle is in contact with the core. In some embodiments,
substantially all of the aromatic monoaldehyde can be reacted such
that the core of the microcapsule contains no free aromatic
monoaldehyde.
In some embodiments, the nanoparticles can have a zeta potential of
5 to 25 millivolts (mV), or 10 to 20 mV, or 15 to 18 mV, or 16 to
17 mV. In some embodiments, zeta potential of the nanoparticles can
be determined at physiological pH, or a pH of 7 to 8, preferably a
pH of 7.4.
In some embodiments, the inorganic nanoparticles are present in an
amount of 1 to 10 weight percent, or 2 to 8 weight percent, of 4 to
6 weight percent, based on the total weight of the
microcapsule.
In some embodiments, the microcapsule has an average diameter of 1
to 50 micrometers, or 1 to 20 micrometers, or 1 to 10 micrometers,
or 2 to 10 micrometers, or 4 to 10 micrometers, or 4 to 9
micrometers.
In an embodiment, the microcapsule comprises a liquid hydrophobic
core comprising peppermint oil and an aromatic monoaldehyde that is
cinnamaldehyde, and a shell comprising amino-functionalized silica
nanoparticles having an average diameter of 140 to 160 nanometers.
The microcapsule includes 1 to 5 volume percent cinnamaldehyde
based on the total volume of the core. In this embodiment, the
microcapsule has an average diameter of 4 to 9 micrometers.
In an embodiment, the microcapsule comprises a liquid hydrophobic
core comprising peppermint oil, and a shell comprising silica
nanoparticles having a first region of functionality and a second
region of functionality. In this embodiment, the first region of
functionality comprises a plurality of amino functional groups
bound to the surface of the nanoparticle. Further in this
embodiment, the second region of functionality comprises a
plurality of ligands formed from the reaction of an amino group
bound to the surface of the nanoparticle and cinnamaldehyde (i.e.,
each ligand is a reaction product formed from the reaction of an
amino group bound to the surface of the nanoparticle and
cinnamaldehyde). The second region of functionality is in contact
with the core of the microcapsule. In this embodiment, the
microcapsule has an average diameter of 4 to 9 micrometers.
In an embodiment, the microcapsule comprises 90 to 99 weight
percent of the liquid hydrophobic core, and 1 to 10 weight percent
of the shell encapsulating the core comprising a plurality of
amino-functionalized silica nanoparticles, wherein the weight
percent of the core and the shell components are based on the total
weight of the microcapsule. In this embodiment, the core comprises
90 to 99 weight percent peppermint oil and 1 to 10 weight percent
cinnamaldehyde, based on the total weight of the core.
Another aspect of the present disclosure is a dispersion comprising
a plurality of stabilized microcapsules. As used herein, "plurality
of microcapsules" refers to a dispersion comprising more than 1
microcapsule, for example more than 10 microcapsules. The
microcapsules can include microcapsules having the above-described
structure and components. In some embodiments, the dispersion can
be in the form of a liquid, a gel, or a paste. In some embodiments,
the dispersion can be in the form of a gel or a paste (e.g., a
toothpaste). In some embodiments, the stabilized microcapsules are
dispersed in an aqueous solution, and the dispersion is in the form
of a liquid. The aqueous solution can comprise water, deionized
water, a buffer (e.g., phosphate buffered saline, phosphate buffer,
and the like), and the like, or a combination thereof. In some
embodiments, the dispersion comprises 1 to 50 volume percent (vol.
%) of the microcapsules, for example 1 to 20 vol. %, for example 5
to 15 vol. %, based on the total volume of the dispersion.
Accordingly, in some embodiments, the dispersion comprises 50 to 99
vol. %, or 80 to 99 vol. %, or 85 to 95 vol. % of the aqueous
solution, based on the total volume of the dispersion.
Depending on the pH of the aqueous solution, the
amino-functionalized inorganic nanoparticles can be converted to
ammonium-functionalized inorganic nanoparticles. Thus in some
embodiments, the microcapsules of the dispersion can have a shell
comprising amino-functionalized inorganic nanoparticles,
ammonium-functionalized inorganic nanoparticles, or a combination
thereof. The pH of the aqueous solution can generally be selected
by a person skilled in the art in order to provide a microcapsule
having the desired surface charge.
In some embodiments, the dispersion excludes a surfactant, for
example the dispersion includes less than 1 weight percent of a
surfactant, or less than 0.1 weight percent of a surfactant,
preferably the dispersion is devoid of a surfactant. In some
embodiments, a crosslinker is excluded from the microcapsules,
where a crosslinker is defined as a compound having two or more
functional groups capable of reaction, for example reaction with a
functional group present on the surface of a nanoparticle.
In some embodiments, the dispersion comprises a plurality of
microcapsules comprising a core comprising an essential oil (e.g.,
peppermint oil) and an aromatic monoaldehyde (e.g., cinnamaldehyde)
and a shell comprising amino-functionalized inorganic nanoparticles
(e.g., silica nanoparticles). When the shell comprises silica, the
silica can be present in an amount of less than or equal to 3
weight percent, or 0.05 to 3 weight percent, or 0.1 to 1.5 weight
percent, or 0.2 to 1.2 weight percent, or 0.3 to 1.2 weight
percent, or 0.5 to 1.2 weight percent, of 0.6 to 1.2 weight
percent, based on the total weight of the dispersion.
The dispersions can be prepared by any method that is generally
known. For example, the dispersion can be prepared by contacting an
aqueous solution comprising the amino-functionalized inorganic
nanoparticles with a hydrophobic liquid (e.g., comprising the
essential oil and the aromatic monoaldehyde) to form a mixture. The
hydrophobic liquid is insoluble in the aqueous solution. Subsequent
to contacting the aqueous solution and the hydrophobic liquid, the
mixture is emulsified, for example using an amalgamator, or any
suitable mixing device. Emulsifying the mixture provides
microcapsules comprising the hydrophobic liquid dispersed in the
aqueous solution, wherein the microcapsules are stabilized by a
shell comprising the amino-functionalized inorganic nanoparticles.
An example of a method for preparing a dispersion according to the
present disclosure is provided in the working examples below.
Another aspect of the present disclosure is a method for treating a
bacterial biofilm. The method comprises contacting the
above-described dispersion with a bacterial biofilm. A "biofilm"
refers to a population of bacteria attached to an inert or living
surface. Thus, biofilms can form on a counter, a table, water
pipes, implants, catheters, cardiac pacemakers, prosthetic joints,
cerebrospinal fluid shunts, endotracheal tubes, and the like. In
some embodiments, the biofilm can be present on a living surface,
for example skin or in a wound, and on teeth (e.g., dental plaque).
Bacteria in a biofilm are enmeshed in an extracellular polymer
matrix, generally a polysaccharide matrix, which holds the bacteria
together in a mass, and firmly attaches the bacterial mass to the
underlying surface. Evidence has shown that biofilms constitute a
significant threat to human health. Wounds and skin lesions are
especially susceptible to bacterial infection.
In some embodiments, the bacterial biofilm can be a gram-negative
bacterial biofilm or a gram-positive bacterial biofilm. In some
embodiments, the bacterial biofilm comprises Escherichia coli
(e.g., E. coli DH5.alpha.), Pseudomonas bacteria (e.g., Pseudomonas
aeruginosa), Staphylococcal bacteria (e.g., Staphylococcal aureus),
Enterobacteriaceae bacteria (e.g., E. cloacae complex),
Streptococcus bacteria, Haemophilus influenzae, Leptospira
interrogans, Legionella bacteria, or a combination thereof.
Contacting the dispersion comprising the microcapsules with a
biofilm can effectively kill bacterial cells present in the
biofilm. Accordingly, the dispersions disclosed herein can be
particularly useful as disinfectants or antimicrobial dispersions.
The contacting can be under conditions effective to treat the
biofilm, for example for a time of 10 minutes to 5 hours, or 1 hour
to 3 hours, and at a temperature of 25 to 37.degree. C. As used
herein, "treating a biofilm" can refer to killing at least 20%, or
at least 40%, or at least 50%, or at least 60%, or at least 80%, or
at least 90% of the bacterial cells present in the biofilm. In some
embodiments, contacting the dispersion with a biofilm can
completely remove the biofilm (i.e., the dispersion is toxic to
greater than 90%, or 99% or 99.9% of the bacterial cells of the
biofilm upon contacting the dispersion with the biofilm).
In some embodiments, contacting the dispersion with a biofilm can
be in the presence of a host cell, for example, mammalian
fibroblasts. In some embodiments, the dispersion is non-toxic to
the host cells, for example, at least 75%, or at least 80%, or at
least 85%, or at least 90%, or at least 95% of the host cells
remain viable after contacting the dispersion. In some embodiments,
the host cells can proliferate in the presence of the dispersion,
for example the number of host cells can increase by at least a
factor of 1.1, or at least a factor of 1.25, or at least a factor
of 1.5 after contacting the dispersion.
In summary, the development of a multimodal antimicrobial delivery
vehicle is reported herein. The nanoparticle stabilized capsules
demonstrate highly effective therapeutic behavior, successfully
eradicating pathogenic biofilm strains of clinical isolates.
Furthermore, the capsules can effectively eliminate a biofilm
infection while promoting fibroblast viability in an in vitro
co-culture model. Future studies will probe capsule performance in
combating in vivo biofilms. These capsules have potential
applications as a general surface disinfectant as well as an
antiseptic for wound treatment. The reactive self-assembly based
strategy provides a promising platform to create effective delivery
vehicles to combat bacterial biofilms.
The microcapsules, dispersions, and methods are further illustrated
by the following non-limiting examples.
EXAMPLES
Silica nanoparticles (SiO.sub.2 NPs) were chosen to stabilize the
emulsions as they are biocompatible, surface functionalization can
be easily introduced, and their diameters can be readily tuned.
See, e.g., Ghouchi Eskandar, N.; Simovic, S.; Prestidge, C. A.
Nanoparticle Coated Submicron Emulsions: Sustained in-Vitro Release
and Improved Dermal Delivery of All-Trans-Retinol. Pharm. Res.
2009, 26, 1764-1775; Stober, W.; Fink, A.; Bohn, E. Controlled
Growth of Monodisperse Silica Spheres in the Micron Size Range.
Journal of Colloid and Interface Science, 1968, 26, 62-69; Rosen,
J. E.; Gu, F. X. Surface Functionalization of Silica Nanoparticles
with Cysteine: A Low-Fouling Zwitterionic Surface. Langmuir 2011,
27, 10507-10513. Control over the size is especially important as
nanomaterials smaller than 70 nm have been shown to readily
penetrate the skin causing detrimental side-effects. See, e.g.,
Rancan, F.; Gao, Q.; Graf, C.; Troppens, S.; Hadam, S.; Hackbarth,
S.; Kembuan, C.; Blume-Peytavi, U.; Ruhl, E.; Lademann, J.; et al.
Skin Penetration and Cellular Uptake of Amorphous Silica
Nanoparticles with Variable Size, Surface Functionalization, and
Colloidal Stability. ACS Nano 2012, 6, 6829-6842; Labouta, H. I.;
Schneider, M. Interaction of Inorganic Nanoparticles with the Skin
Barrier: Current Status and Critical Review. Nanomedicine:
Nanotechnology, Biology, and Medicine, 2013, 9, 39-54; Nabeshi, H.;
Yoshikawa, T.; Matsuyama, K.; Nakazato, Y.; Matsuo, K.; Arimori,
A.; Isobe, M.; Tochigi, S.; Kondoh, S.; Hirai, T.; et al. Systemic
Distribution, Nuclear Entry and Cytotoxicity of Amorphous
Nanosilica Following Topical Application. Biomaterials 2011, 32,
2713-2724. Therefore, we synthesized cationic amino-functionalized
SiO.sub.2 NPs with an average diameter of .about.150 nm, as shown
in FIG. 1-3. FIG. 1 shows a transmission electron micrograph (TEM)
image of silica nanoparticles having an average diameter of
152.+-.15 nm (scale bar is 500 nm). The inset shows a histogram of
the measured nanoparticle diameters. FIG. 2 and FIG. 3 show the
results from dynamic light scattering (DLS) and zeta potential
measurements, performed using a Malvern Zetasizer Nano ZS. FIG. 2
shows that average diameter of the silica nanoparticles in MilliQ
water was 171.5.+-.2.8 nm. FIG. 3 shows that zeta potential of the
silica nanoparticles in 5 millimolar (mM) sodium phosphate buffer
at pH 7.4 was 16.7.+-.0.1 mV.
Antimicrobial capsules were generated using a Pickering emulsion
template as shown in FIG. 4. As shown in FIG. 4, peppermint oil
with dissolved cinnamaldehyde is emulsified into an aqueous
suspension of amine functionalized silica nanoparticles.
Cinnamaldehyde within the oil reacts with the amines on the
nanoparticles at the oil/water interface to create a multimodal
delivery vehicle. Capsules can interact with a biofilm through
electrostatic complementarity. Capsules release their payload
disrupting the biofilm, eliminating the bacteria. Capsules were
created by emulsifying either peppermint oil or a mixture of
cinnamaldehyde dissolved in peppermint oil into MilliQ H.sub.2O
adjusted to a pH of 10 containing the nanoparticles. The
nanoparticles self-assemble at the oil/water interface to stabilize
the peppermint oil droplets. Surface amines on the nanoparticles
then react with the cinnamaldehyde within the oil phase.
Silica loadings in the aqueous phase were varied to determine the
amount needed to minimize capsule dispersity. At loadings of 1.2
wt. % SiO.sub.2 NPs or greater, capsules were found to have a
minimum dispersity and therefore this amount was chosen for all
further studies. The effect of SiO.sub.2 NP loading is shown in the
optical micrographs of FIG. 5. Optical images of the capsules were
taken on an Olympus IX51 microscope. Capsules were generated using
aqueous phases comprising a) 0.3 wt. % silica (average
diameter=6.1.+-.3.0 .mu.m), b) 0.6 wt. % silica (average
diameter=7.1.+-.3.0 .mu.m), c) 0.9 wt. % silica (average
diameter=6.7.+-.2.1 .mu.m), d) 1.2 wt. % silica (average
diameter=6.5.+-.1.7 .mu.m), and e) 1.5 wt. % silica (average
diameter=6.6.+-.2.1 .mu.m), respectively. The inset of each
micrograph shows the histogram of the capsule diameter
measurements. FIG. 5 also shows a box plot of the capsules at
various silica loadings demonstrating capsule dispersity minimizes
with increasing wt. % of silica nanoparticles.
It was also observed that capsules generated with higher than 5%
v/v cinnamaldehyde were unstable which corresponds to 52-fold
excess of cinnamaldehyde to available amines on the nanoparticle
surface. The peppermint oil based capsules ("P-Cap") and capsules
containing 5% v/v of cinnamaldehyde dissolved in peppermint oil
("CP-Cap") were found to have average diameters of 6.8.+-.1.9
micrometers (.mu.m) and 6.7.+-.1.9 .mu.m, respectively, as shown in
the optical microscopy images of FIG. 6. FIG. 6 shows P-Cap had an
average diameter of 6.8.+-.1.9 .mu.m (a) and CP-Cap had an average
diameter of 6.7.+-.1.9 .mu.m (b). The insets are histograms of the
capsule diameter measurements.
Various techniques were used to probe the
cinnamaldehyde-nanoparticle interaction, including confocal
microscopy, X-ray photoelectron spectroscopy (XPS), attenuated
total reflection Fourier transform infrared spectroscopy
(ATR-FTIR), and contact angle goniometry. Reactive molecules within
the oil core of Pickering emulsions have been previously
demonstrated to affect capsule morphologies by modulating the
hydrophobicity of the nanoparticles. See, e.g., Duncan, B.; Landis,
R. F.; Jerri, H. a; Normand, V.; Benczedi, D.; Ouali, L.; Rotello,
V. M. Hybrid Organic-Inorganic Colloidal Composite "Sponges" via
Internal Crosslinking. Small 2015, 11, 1302-1309; Williams, M.;
Warren, N. J.; Fielding, L. A.; Armes, S. P.; Verstraete, P.;
Smets, J. Preparation of Double Emulsions Using Hybrid
Polymer/Silica Particles: New Pickering Emulsifiers with Adjustable
Surface Wettability. ACS Appl. Mater. Interfaces 2014, 6,
20919-20927. To determine if structural reorganization occurs with
our mixed oil system, capsules were generated using a Nile red
loaded oil core and nanoparticles possessing cores labeled with
fluorescein isothiocyanate (FITC). As shown in FIG. 7, both
capsules with and without cinnamaldehyde possess core-shell
morphologies. This result indicates that the 5% v/v loading of
cinnamaldehyde into the peppermint oil does not alter the capsule
structure
XPS and ATR-FTIR were used to elucidate the reactivity of the
nanoparticles with the dissolved cinnamaldehyde of the capsules.
Prior to analysis, CP-Caps were disrupted with ethanol,
centrifuged, and lyophilized to remove any adsorbed cinnamaldehyde.
The Schiff base of 3-aminopropyltriethoxysilane and cinnamaldehyde
was also synthesized for comparison. As shown in FIG. 7(c), the
SiO.sub.2 NPs showed two chemically distinct species with a lower
binding energy (BE) component at ca. 399.5 eV and a higher BE
component at ca. 401.8 eV. These correspond to amine (--NH--) and
protonated amine (NH3+) present on the surface of SiO.sub.2 NPs
that is consistent with previously reported values. See, e.g., Tan,
K.; Tan, B.; Kang, E.; Neoh, K. X-Ray Photoelectron Spectroscopy
Studies of the Chemical Structure of Polyaniline. Phys. Rev. B,
1989, 39, 8070-8073. The N 1s spectra of CP-Cap shows three
distinct chemical species. In addition to the two N 1s BE
components observed in the SiO2 NPs, a new peak centered at ca.
400.1 eV indicates the formation of an imine functional
(--C.dbd.N--) group which corroborates well with literature values.
See, e.g., Ricci, M.; Trinquecoste, M.; Auguste, F.; Canet, R.;
Delhaes, P.; Guimon, C.; Pfister-Guillouzo, G.; Nysten, B.; Issi,
J. P. Relationship between the Structural Organization and the
Physical Properties of PECVD Nitrogenated Carbons. J. Mater. Res.,
1993, 8, 480-488. The N 1s spectra from the synthesized Schiff base
(FIG. 8) showed a single chemically distinct N 1s species centered
at ca. 400.2 eV, which corresponds to the imine functional group
(--C.dbd.N--). Similarly, the chemically distinct species of the C
1s spectra obtained from CP-Cap matches well with the synthesized
Schiff base further providing evidence on the covalent linkage of
the amine and cinnamaldehyde (FIG. 8). Additionally, the Si 2p and
O 1s peak shows typical BEs centered at ca. 103.2 eV and 532.6 eV,
respectively that matches with reported values for SiO.sub.2 NPs.
See, e.g., Ramanathan, R.; Campbell, J. L.; Soni, S. K.; Bhargava,
S. K.; Bansal, V. Cationic Amino Acids Specific Biomimetic
Silicification in Ionic Liquid: A Quest to Understand the Formation
of 3-D Structures in Diatoms. PLoS One 2011, 6.
ATR-FTIR analysis further supported the formation of the
cinnamaldehyde Schiff base, as shown in FIG. 9. IR was performed on
a Bruker Alpha FTIR spectrophotometer fitted with a platinum ATR
QuickSnap sampling module. Cinnamaldehyde was analyzed as a pure
liquid. The silica nanoparticles and Schiff base were analyzed as
neat solids. The CP-Caps were dissolved in ethanol, sonicated, and
centrifuged three times to remove the oil phase. The CP-Cap pellet
was then freeze dried in order to obtain a dry solid that was
analyzed. Cinnamaldehyde displayed characteristic peaks at 1667
cm.sup.-1 and 1624 cm.sup.-1 attributed to the C.dbd.O and C.dbd.C
frequencies. The Schiff base possessed peaks at 1681 cm.sup.-1 and
1633 cm.sup.-1 attributed to C.dbd.N and C.dbd.C bonds,
respectively. The Schiff base also displayed peaks at 1102
cm.sup.-1 and 1006 cm.sup.-1 attributed to the Si--O and Si-OEt
frequencies. Amine-functionalized silica nanoparticles showed
characteristic peaks at 1080 cm.sup.-1 and 947 cm.sup.-1 attributed
to the Si--O and Si--OH frequencies. CP-Caps displayed peaks at
frequencies similar to the SiO.sub.2 NPs and the Schiff base
confirming the formation of the Schiff base complex on the
nanoparticles.
An in situ covalent reaction of the primary amine groups on the
nanoparticles with cinnamaldehyde should alter the hydrophobicity
of the nanoparticle surface improving the stabilization behavior of
the Pickering emulsifiers. See, e.g., Pieranski, P. Two-Dimensional
Interfacial Colloidal Crystals. Phys. Rev. Lett. 1980, 45, 569-572.
Contact angle goniometry was used to measure the change in
nanoparticle hydrophobicity, as shown in FIG. 10. Nanoparticles
were deposited onto silicon wafers and briefly incubated in
dichloromethane solutions with varying amounts of dissolved
cinnamaldehyde. The surfaces were then rinsed with dichloromethane,
dried, and the water contact angles were obtained. FIG. 10 shows
that as the percentage of cinnamaldehyde by volume increases from
0% to 5% %, the water contact angle of the nanoparticles increases
from 31.degree. to 49.degree.. This increase in water contact
angle, taken together with the XPS data, the ATR-FTIR data, and
confocal images, indicates that the inclusion of cinnamaldehyde
within the peppermint oil core generates a distinct,
multi-component capsule structure.
Biofilms produce extracellular polymeric substances that prevent
effective delivery of therapeutics. See, e.g., Hurdle, J. G.;
O'Neill, A. J.; Chopra, I.; Lee, R. E. Targeting Bacterial Membrane
Function: An Underexploited Mechanism for Treating Persistent
Infections. Nat. Rev. Microbiol. 2011, 9, 62-75. Having established
that the capsules have core-shell morphologies and the
cinnamaldehyde is successfully incorporated into the capsules, we
set out to determine whether these capsules could effectively
penetrate into biofilms. Using fluorescently labeled nanoparticles
to track the delivery of the emulsions, we treated biofilms from E.
coli that had been modified to express E2-Crimson, a far-red
fluorescent protein. As shown in FIG. 11, both P-Cap and CP-Cap
diffuse into the biofilm matrix and efficiently disperse throughout
the biofilm whereas the unassembled nanoparticles displayed minimal
penetration. These data indicate the capsules deliver their payload
in a burst release fashion and that both the oil core and
nanoparticle shell are operative for effective delivery.
Next, we investigated the therapeutic behavior of the capsules
against established bacterial biofilms. One laboratory strain, E.
coli DH5.alpha., and 3 pathogenic bacteria strains of clinical
isolates, P. aeruginosa (CD-1006), S. aureus (CD-489, a
methicillin-resistant strain), and E. cloacae complex (CD-1412),
were chosen to test our system. As shown in FIG. 12, both the
CP-Cap and P-Cap vehicles effectively were able to kill bacteria
cells in all four biofilms, with CP-Cap possessing greater
activity. The capsules demonstrated a dramatically enhanced
efficacy compared with the unencapsulated oil, supporting the
hypothesis that the cationic nanoparticle shell of the capsules
increases interaction with the biofilms. See, e.g., Li, X.; Yeh,
Y.; Giri, K.; Mout, R.; Landis, R. F.; Prakash, Y. S.; Rotello, V.
M. Control of Nanoparticle Penetration into Biofilms through
Surface Design. Chem. Commun. (Camb). 2015, 51, 282-285. In
addition, the acidic pH of the biofilm environment should promote
the hydrolysis of Schiff bases, enhancing the sustained release of
cinnamaldehyde. See, e.g., Harrison, J. J.; Ceri, H.; Turner, R. J.
Multimetal Resistance and Tolerance in Microbial Biofilms. Nat.
Rev. Microbiol. 2007, 5, 928-938. These capsules were able to treat
both Gram negative (E. coli, P. aeruginosa, and E. cloacae complex)
and Gram positive (S. aureus) bacteria. Notably, the capsules
demonstrated a similar efficacy against the multi-drug resistant S.
aureus stain when compared to the non-resistant strains, supporting
that these capsules present a viable treatment alternative to
traditional antibiotics.
Biofilm infections within wounds interfere with the ability of the
host to regenerate damaged tissue. See, e.g., Roy, S.; Elgharably,
H.; Sinha, M.; Ganesh, K.; Chaney, S.; Mann, E.; Miller, C.;
Khanna, S.; Bergdall, V. K.; Powell, H. M.; et al. Mixed-Species
Biofilm Compromises Wound Healing by Disrupting Epidermal Barrier
Function. J. Pathol. 2014, 233, 331-343. Fibroblasts in particular
play a vital role in the wound healing process, helping to close
the injury and redevelop the extracellular matrix within the skin.
See, e.g., Watt, F. M. Mammalian Skin Cell Biology: At the
Interface between Laboratory and Clinic. Science 2014, 346,
937-940; Sun, B. K.; Siprashvili, Z.; Khavari, P. A. Advances in
Skin Grafting and Treatment of Cutaneous Wounds. Science 2014, 346,
941-945. We used an in vitro co-culture model comprised of
mammalian fibroblasts and a biofilm to determine whether our
capsules could successfully treat a biofilm in the presence of host
cells. See, e.g., Li, X.; Kong, H.; Mout, R.; Saha, K.; Moyano, D.
F.; Robinson, S. M.; Rana, S.; Zhang, X.; Riley, M. A.; Rotello, V.
M. Rapid Identification of Bacterial Biofilms and Biofilm Wound
Models Using a Multichannel Nanosensor. ACS Nano 2014, 8,
12014-12019. E. coli DH5.alpha. bacteria were seeded with a
confluent NIH 3T3 fibroblast cell monolayer overnight to generate
biofilms prior to treatment. The co-cultures were treated with
capsules for 3 hours, washed, and the viabilities of both
fibroblasts and bacteria were measured. As shown in FIG. 13, CP-Cap
effectively treated the biofilm infection whereas P-Cap and the
controls did not. The capsule structure also prevented the toxic
effects shown by the unencapsulated peppermint oil to the
fibroblasts. Notably, CP-Cap enhanced 3T3 cell growth in agreement
with studies that cinnamaldehyde can promote insulin-like growth
factor-I signaling, increasing cell proliferation. See, e.g.,
Takasao, N.; Tsuji-Naito, K.; Ishikura, S.; Tamura, A.; Akagawa, M.
Cinnamon Extract Promotes Type I Collagen Biosynthesis via
Activation of IGF-I Signaling in Human Dermal Fibroblasts. J.
Agric. Food Chem. 2012, 60, 1193-1200.
Experimental Details Follow.
Materials. All reagents/materials were purchased from Fisher
Scientific and used as received. Boron-doped Si wafers were
purchased from WRS Materials. NIH-3T3 cells (ATCC CRL-1658) were
purchased from ATCC. Dulbecco's Modified Eagle's Medium (DMEM)
(DMEM; ATCC 30-2002) and fetal bovine serum (Fisher Scientific,
SH3007103) were used in cell culture. Pierce LDH Cytotoxicity Assay
Kit was purchased from Fisher Scientific.
Silica nanoparticles were synthesized according to the reported
procedure. See, e.g., Duncan, B.; Landis, R. F.; Jerri, H. A;
Normand, V.; Benczedi, D.; Ouali, L.; Rotello, V. M. Hybrid
Organic-Inorganic Colloidal Composite "Sponges" via Internal
Crosslinking. Small 2015, 11, 1302-1309; Stober, W.; Fink, A.;
Bohn, E. Controlled Growth of Monodisperse Silica Spheres in the
Micron Size Range. J. Colloid Interface Sci. 1968, 26, 62-69.
Briefly, to synthesize 150 nanometer amine-functionalized silica
nanoparticles, 24 milliliters of ammonium hydroxide were added to
300 milliliters of absolute ethanol and stirred for five minutes in
a 500 milliliters round-bottom flask. Then 12 milliliters of
tetraethyl orthosilicate were added to the reaction flask and
stirred overnight at room temperature. To functionalize the surface
of the nanoparticles, 1.22 milliliters of 3-aminopropyl
triethoxysilane were added and the reaction was stirred for an
additional 24 hours. The nanoparticles were purified by
centrifuging and re-dispersing in water and ethanol (3 times
each).
Synthesis of fluorescein labeled nanoparticles (Santra, S.;
Liesenfeld, B.; Bertolino, C.; Dutta, D.; Cao, Z.; Tan, W.;
Moudgil, B. M.; Mericle, R. A. Fluorescence Lifetime Measurements
to Determine the Core--shell Nanostructure of FITC-Doped Silica
Nanoparticles: An Optical Approach to Evaluate Nanoparticle
Photostability. J. Lumin. 2006, 117, 75-82) was performed following
a similar method wherein 5.25 milligrams of fluorescein
isothiocyanate were reacted with 69.0 milligrams of 3-aminopropyl
triethoxysilane in one milliliter of absolute ethanol under
nitrogen overnight. This fluorescent conjugate mixture was added to
the nanoparticle reaction solution 5 minutes after the addition of
tetraethyl orthosilicate. The procedure then proceeded as described
above.
Stock capsules solutions were prepared in 1.5 milliliter Eppendorf
tubes. To prepare the stock P-Cap emulsions, 300 microliters
(.mu.L) of peppermint oil was added to 1.2 milliliters of a 1.2%
wt. solution of SiO.sub.2 NPs in MilliQ H.sub.2O adjusted to pH 10
and was emulsified in an amalgamator for 50 seconds. To prepare the
stock CP-Cap emulsions, 15 .mu.L of cinnamaldehyde was dissolved in
285 .mu.L of peppermint oil prior to emulsification as described.
The emulsions were allowed to rest overnight prior to use.
X-ray photoelectron spectroscopy (XPS) samples were prepared by
drop-casting the sample on a 100 nanometer gold-coated silicon
substrate. XPS measurements were carried out using Physical
Electronics Quantum 2000 spectrometer at a pressure below
1.times.10.sup.-9 Torr. The survey scan, C 1s, N 1s, O 1s and Si 2p
core level spectra for all samples were recorded with
un-monochromatized Al K.alpha. radiation (photon energy of 1486.6
eV) at a pass energy of 46.95 eV and electron takeoff angle of
15.degree.. The overall resolution was 0.2 eV for the XPS
measurements. Chemically distinct species were resolved using a
Gaussian-Lorentzian function with non-linear least-square fitting
procedure. All XPS spectra were background corrected using the
Shirley algorithm and aligning the elemental binding energies to
the adventitious carbon (C1s) binding energy of 284.6 eV.
Contact angle goniometry samples were prepared by immersing a clean
silicon wafer (1 centimeter.times.1 centimeter) into 1 milliliter
of a 1.2% wt. solution of SiO.sub.2 NPs in MilliQ H.sub.2O adjusted
to pH 10 for 5 minutes. Wafers were then washed with MilliQ
H.sub.2O to removed excess nanoparticles and dried under a N.sub.2
stream. Samples were then incubated in 1 milliliter solutions of
dichloromethane with varying amounts (0, 1, 2, 5 volume percent) of
dissolved cinnamaldehyde for 5 minutes. Wafers were then washed
with dichloromethane and dried under a nitrogen stream. Static
water contact angles were measured using a VCA Optima surface
analysis/goniometry system with water droplets size of 2 .mu.L.
Biofilm Formation
Biofilms were grown as previously reported. See, e.g., Li, X.;
Kong, H.; Mout, R.; Saha, K.; Moyano, D. F.; Robinson, S. M.; Rana,
S.; Zhang, X.; Riley, M. A.; Rotello, V. M. Rapid Identification of
Bacterial Biofilms and Biofilm Wound Models Using a Multichannel
Nanosensor. ACS Nano 2014, 8, 12014-12019. Bacteria were inoculated
in lysogeny broth (LB) medium at 37.degree. C. until stationary
phase. The cultures were then harvested by centrifugation and
washed with 0.85% sodium chloride solution three times.
Concentrations of resuspended bacterial solution were determined by
optical density measured at 600 nm. LB medium was supplemented with
0.1% glucose, 1 mM magnesium sulfate, 0.15 M ammonium sulfate, and
34 mM citrate and buffered to pH 7 to ensure bacterial adherence to
the microplate. Seeding solutions were then made in this modified
LB medium to reach an OD.sub.600 of 0.1. A 100 .mu.L amount of the
seeding solutions was added to each well of the 96 well microplate.
The plates were covered and incubated at room temperature under
static conditions for 1 day.
A 2 volume percent emulsion stock solution was made by diluting the
generated capsules into LB medium. The stock solution was then
diluted to the desired level and incubated with the biofilms for 3
hours. Biofilms were washed with phosphate buffer saline (PBS)
three times and viability was determined using an Alamar Blue
assay. See, e.g., Margarida Pereira, A.; Cristina Abreu, A.;
Simoes, M. Action of Kanamycin Against Single and Dual Species
Biofilms of Escherichia Coli and Staphylococcus Aureus. J.
Microbiol. Res. 2012, 2, 84-88. Modified LB medium without bacteria
was used as a negative control.
Biofilm-3T3 Fibroblast Cell Co-Culture
Co-culture was performed as previously described. See, e.g.,
Takasao, N.; Tsuji-Naito, K.; Ishikura, S.; Tamura, A.; Akagawa, M.
Cinnamon Extract Promotes Type I Collagen Biosynthesis via
Activation of IGF-I Signaling in Human Dermal Fibroblasts. J.
Agric. Food Chem. 2012, 60, 1193-1200. Briefly, a total of 20,000
NIH 3T3 (ATCC CRL-1658) cells were cultured in Dulbecco's modified
Eagle medium (DMEM; ATCC 30-2002) with 10% bovine calf serum and 1%
antibiotics at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. Cells were kept for 24 h to reach a confluent monolayer.
Bacteria were inoculated and harvested as described above, and
seeding solutions were made in buffered DMEM supplemented with
glucose to reach an OD.sub.600 of 0.1. Old medium was removed from
3T3 cells followed by addition of 100 .mu.L of seeding solution.
The co-cultures were then stored in a box with damp paper towels at
37.degree. C. overnight without shaking.
Testing solutions at different concentrations were made by diluting
capsules into DMEM prior to use. Media was removed from co-culture,
replaced with testing solutions, and incubated for 3 hours at
37.degree. C. Co-cultures were then analyzed using a LDH
cytotoxicity assay to determine mammalian cell viability according
the manufacturer's instructions. See, e.g., Decker, T.;
Lohmann-Matthes, M. L. A Quick and Simple Method for the
Quantitation of Lactate Dehydrogenase Release in Measurements of
Cellular Cytotoxicity and Tumor Necrosis Factor (TNF) Activity. J.
Immunol. Methods 1988, 115, 61-69. To determine bacteria viability
in biofilms, the testing solutions were removed and co-cultures
were washed with PBS. Fresh PBS was then added to disperse
remaining bacteria from biofilms in co-culture by sonication for 20
minutes and mixing with pipette. The solutions containing dispersed
bacteria were then plated onto agar plates and colony forming units
were counted after incubation at 37.degree. C. overnight.
The microcapsules, dispersions and methods of the present
disclosure include at least the following embodiments.
Embodiment 1
A stabilized microcapsule comprising a liquid hydrophobic core
comprising an essential oil and an aromatic monoaldehyde; and a
shell encapsulating the core, the shell comprising a plurality of
amino-functionalized inorganic nanoparticles.
Embodiment 1a
A stabilized microcapsule comprising, a liquid hydrophobic core
comprising an essential oil; and a shell encapsulating the core,
the shell comprising a plurality of amino-functionalized inorganic
nanoparticles, wherein the amino-functionalized inorganic
nanoparticles further comprise a reaction product formed from
reaction of an amino-functionalized inorganic nanoparticle and an
aromatic monoaldehyde covalently bound to a surface of the
nanoparticle.
Embodiment 2
The microcapsule of embodiment 1 or 1a, wherein the inorganic
nanoparticles are metal nanoparticles, metal oxide nanoparticles,
or a combination thereof, and wherein the nanoparticles have one or
more dimensions of less than 1000 nanometers.
Embodiment 3
The microcapsule of any of embodiments 1 to 2, wherein the
inorganic nanoparticles comprise silica, titanium dioxide, or a
combination thereof.
Embodiment 4
The microcapsule of any of embodiments 1 to 3, wherein the
inorganic nanoparticles comprise silica.
Embodiment 5
The microcapsule of any of embodiments 1 to 4, wherein the
inorganic nanoparticles have an average diameter of 100 to 250
nanometers.
Embodiment 6
The microcapsule of any of embodiments 1 to 5, wherein the
essential oil is selected from the group consisting of peppermint
oil, oregano oil, thymol, menthol, methyl salicylate, eucalyptol,
carvacrol, camphor, anethole, carvone, eugenol, isoeugenol,
limonene, osimen, n-decyl alcohol, citronel, a-salpineol, methyl
acetate, citronellyl acetate, methyl eugenol, cineol, linalool,
ethyl linalaol, safrola vanillin, spearmint oil, lemon oil, orange
oil, sage oil, rosemary oil, cinnamon oil, pimento oil, laurel oil,
cedar leaf oil, clove oil, cilantro oil, coriander oil, or a
combination thereof.
Embodiment 7
The microcapsule of any of embodiments 1 to 6, wherein the
essential oil comprises peppermint oil.
Embodiment 8
The microcapsule of any of embodiments 1 to 7, wherein the aromatic
monoaldehyde is cinnamaldehyde.
Embodiment 9
The microcapsule of any of embodiments 1 to 8, wherein the
amino-functionalized inorganic nanoparticles further comprise a
reaction product covalently bound to a surface of the nanoparticle,
wherein the reaction product is formed from reaction of an
amino-functionalized inorganic nanoparticle and one or more
aromatic monoaldehydes.
Embodiment 10
The microcapsule of any of embodiments 1 to 9, comprising 0.01 to
10 volume percent of the aromatic monoaldehyde, based on the total
volume of the essential oil and the aromatic monoaldehyde.
Embodiment 11
The microcapsule of any of embodiments 1 to 10, comprising 1 to 10
weight percent of the inorganic nanoparticles, based on the total
weight of the microcapsule.
Embodiment 12
The microcapsule of any of embodiments 1 to 11, wherein the
microcapsule has an average diameter of 1 to 20 micrometers.
Embodiment 13
The microcapsule of any of embodiments 1 to 12, wherein the
essential oil comprises peppermint oil; the aromatic monoaldehyde
is cinnamaldehyde; the microcapsule comprises 1 to 5 volume percent
cinnamaldehyde, based on the total volume of the essential oil and
the aromatic monoaldehyde; the inorganic nanoparticles are silica
nanoparticles having an average diameter of 140 to 160 nanometers;
and the microcapsule has an average diameter of 4 to 9
micrometers.
Embodiment 14
The microcapsule of any of embodiments 1 to 13, comprising, based
on the total weight of the microcapsule, 90 to 99 weight percent of
the liquid hydrophobic core; and 1 to 10 weight percent of a shell
encapsulating the core; wherein the liquid hydrophobic core
comprises, based on the total weight of the core, 90 to 99 weight
percent peppermint oil; and 1 to 10 weight percent
cinnamaldehyde.
Embodiment 15
A dispersion comprising a plurality of stabilized microcapsules
according to any of embodiments 1 to 14.
Embodiment 16
The dispersion of embodiment 15, wherein the stabilized
microcapsules are dispersed in an aqueous solution.
Embodiment 17
The dispersion of embodiment 15 or 16, wherein each microcapsule
comprises a shell comprising silica nanoparticles; and the silica
is present in an amount of less than or equal to 3 weight percent,
based on the total weight of the dispersion.
Embodiment 18
A method of treating a bacterial biofilm, the method comprising,
contacting the dispersion of any of embodiments 15 to 17 with a
bacterial biofilm.
Embodiment 19
The method of embodiment 18, wherein the bacterial biofilm
comprises Escherichia coli, Pseudomonas bacteria, Staphylococcal
bacteria, Enterobacteriaceae bacteria, Streptococcus bacteria,
Haemophilus influenzae, Leptospira interrogans, Legionella
bacteria, or a combination thereof.
Embodiment 20
The method of embodiment 18 or 19, wherein the contacting is in the
presence of a host cell, wherein the dispersion is non-toxic to the
host cell.
This written description uses examples to disclose the invention,
including the best mode, and also to enable any person skilled in
the art to make and use the invention. The patentable scope of the
invention is defined by the claims, and may include other examples
that occur to those skilled in the art. Such other examples are
intended to be within the scope of the claims if they have
structural elements that do not differ from the literal language of
the claims, or if they include equivalent structural elements with
insubstantial differences from the literal language of the
claims.
All cited patents, patent applications, and other references are
incorporated herein by reference in their entirety, including
priority U.S. Patent Application No. 62/208,114, filed Aug. 21,
2015. However, if a term in the present application contradicts or
conflicts with a term in the incorporated reference, the term from
the present application takes precedence over the conflicting term
from the incorporated reference.
All ranges disclosed herein are inclusive of the endpoints, and the
endpoints are independently combinable with each other. Each range
disclosed herein constitutes a disclosure of any point or sub-range
lying within the disclosed range. "Or" means "and/or".
"Combination" is inclusive of blends, mixtures, alloys, reaction
products, and the like.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Further, it should further be
noted that the terms "first," "second," and the like herein do not
denote any order, quantity, or importance, but rather are used to
distinguish one element from another.
* * * * *